Fluorescence microscopy has the unique capacity to probe both static and live processes with great specificity to link the dynamics and/or localization of molecular and cellular components with their function. Recently, a new microscope platform, OMX, was designed to acquire sub-second four-dimensional (4D) multi-color in vivo data with a dual functionality to attain sub-diffraction structured illumination (SI) imaging of fixed samples. Over the last two years, this first generation OMX microscope at UCSF has been converted from a dedicated development microscope to a production microscope open to projects from within UCSF and from the outside academic community. As a result of more general use, users identified several major desired improvements which, if they could be made to the OMX microscope, would vastly expand their ability to attain their research goals. The first update is to increase the time over which biological processes can be observed in their unperturbed natural state. Phototoxicity is a major limitation in live microscopy, inducin morphological changes, delays in cell progression and cell death. Introduction of pulsed lasers to reduce the excitation light to microsecond exposures rather than the current millisecond limits will permit far lower photon doses to be achieved, allowing cells to be imaged for much longer periods of time. The second update is to use recent improvements in camera and stage technology to increase the speed and stability of 3D structural illumination data acquisition. This update will have the added benefit of permitting in vivo 3D SI. Currently the quality and throughput of 3D SI microscopy is severely compromised by drift between consecutive sections in a 3D image stack. In this application, we seek funds to revolutionize the technological base of the OMX microscope for far faster and more stable data acquisition for both live and SI imaging. The proposed enhancements include 1) upgrading our lasers to pulsed lasers to achieve microsecond exposure times; 2) incorporating a sCMOS camera with faster acquisition rates, which in combination with the pulsed lasers will permit a 3D SI data stack to be acquired in 3 seconds rather than the current 13 minutes; 3) replacing our current xyz stage that introduces thermally induced drifts with a more modern stage to improve the stability, speed and depth of SI data acquisitions; and 4) upgrading the computer that will control the new stage motors to one with PCI/PCI(e) slots. These technological advances will benefit a great many biomedical research projects funded by NIH and will be of vital importance in elucidating the basic biological processes underlying many human diseases. PUBLIC HEALTH RELEVANCE: Studies of the dynamic processes occurring in living organisms in a non-perturbed setting is of vital importance in understanding the basic mechanisms of the biological processes underlying many human diseases. Rapid three-dimensional in vivo and super-resolution structured illumination imaging have become powerful new techniques in monitoring the changes that occur in the cell. This application will 1) greatly extend the ability of this technology to follow a biological process through its entire course without perturbation of its natural state and 2) enable super-resolution microscopy on live samples heretofore could only be examined in non-living specimens.